[Expression of EGF and SP domain proteins of MASP-2 and preparation of their polyclonal antibodies].

Aim: To clone and prokaryotically express the EGF and SP domain proteins of human mannan-binding lectin associated serine protease-2 (MASP-2), and prepare their polyclonal antibodies and analyze their immunogenicity.

Methods: The cDNA of human MASP-2 was transcripted reversely from total RNA extracted from human fetal liver tissue, and then EGF and SP domain genes of MASP-2 were amplified from the cDNA by PCR. The EGF and SP fragment domain genes were cloned into pGEX-6P-2 expression vector and induced by IPTG to express GST fusion protein. The BALB/c mice were immunized with the purified fusion proteins to generate polyclonal antibodies. The specificity of the polyclonal antibodies was analysed with Western blotting, and the titer was determined by indirect ELISA.

Results: We successfully constructed the recombinant pGEX-6P-2-EGF and pGEX-6P-2-SP and induced the expressions of the GST-EGF and GST-SP fusion proteins. The specificity of the polyclonal antibodies was higher, and had no cross reaction with other proteins. Indirect ELISA showed that the titer of anti-EGF antibody was more than 1:32 000 and anti-SP antibody was more than 1:40 000.

Conclusion: The polyclonal antibodies of the EGF and SP domain proteins we successfully obtained were of higher specificity and titer, which provides the basis for further research on MASP-2.